Point-of-care (POC) testing is an analytical method conducted outside the central hospital and/or laboratory using devices that can instantly interpret the results. With the increasing threat of accelerated epidemic-to-pandemic transitions of new or reemerging infectious disease outbreaks owing to globalization, decentralizing diagnostic testing at frontline clinical settings could facilitate earlier implementations of public health responses to contain and mitigate such events. In the developing countries where high infectious disease burden is compounded by diagnostic challenges due to poor clinical laboratory infrastructure and cost constraints, the potential utility for POC testing is even greater.
Although microbial culture, microscopy examination and metabolites biochemical test are still used in some scenarios, today the majority of POC device and assay development are different types of PCR (polymerase chain reaction, such as nested PCR, real-time PCR and digital PCR) based methods. PCR is an enzyme-driven process for amplifying short regions of DNA in vitro where millions of DNA copies are created by cycling among different temperatures to allow repeating steps (denaturation, annealing and elongation) of DNA replication to take place. Despite the simplicity and amplification power of PCR chemistry, requirement of precision thermal cycling among two or three temperature zones during the reaction is an inevitable drawback for PCR development in POC diagnostics.
Above mentioned limitations of the PCR reaction stimulates the development of alternative isothermal amplification methods. Instead of using thermal cycling, isothermal amplification relies on proteins that use in vivo mechanisms of DNA/RNA synthesis and dominated by enzyme activity. Therefore, miniaturize isothermal system has advantages of simple design and extremely low energy consumption. Today, various isothermal based amplification methods in terms of assay complexity (multiple enzymes or primers), acceptable detection sensitivity, and specificity have been developed, including nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and nicking enzyme amplification reaction (NEAR).
As a relative new solution, the platform design of isothermal method lags behind its biochemical technique development. Because isothermal amplification bears higher tolerance on the sample purity, most of commercial isothermal platforms focus on creating a stable temperature environment and detection methods with middle and high throughput. The sample preparation, however, is carried out either using external equipment or manual operation. On the other hand, with the increasingly demand of simultaneously detection of multi targets, the ability of multiplexing is inevitable for isothermal based POC platforms. Unfortunately, there is no all-in-one commercial platform being able to detect multiple targets with isothermal amplification methods.
Thus, there is a need of providing an all-in-one nucleic acid analysis apparatus with isothermal based amplification to overcome the drawbacks of the prior arts.